Synergism of a mixed diet of Myzus persicae and egg of Ephestia kuehniella on fitness of the predator Nabis stenoferus

Nabis stenoferus is a zoophytophagous predator that lives in grasslands around agricultural fields. It is a candidate biological control agent for use via augmentation or conservation. To find a suitable food source for mass-rearing and to better understand this predator’s biology, we compared the life history characteristics of N. stenoferus under the three different diets: aphids only (Myzus persicae), moth eggs only (Ephestia kuehniella), or a mixed diet of aphids and moth eggs. Interestingly, when only aphids were supplied, N. stenoferus developed to the adult stage but lacked normal levels of fecundity. There was a significant synergism of the mixed diet on N. stenoferus fitness in both the immature and adult stages, i.e., a 13% reduction in the nymphal developmental period and an 87.3-fold increase in fecundity, compared to aphid-only diet. Furthermore, the intrinsic rate of increase was significantly higher for the mixed diet (0.139) than either aphids only (0.022) or moth eggs only (0.097). These results show that M. persicae alone is not a complete diet for the mass-rearing of N. stenoferus, whereas this aphid can be a supplementary food when combined with E. kuehniella eggs. Implications and applications of these findings for biological control are discussed.

the population potential of an insect reared under specific conditions that can be calculated from data on developmental time, fecundity, longevity, sex ratio, and survivorship 25,26 . Population parameters derived from life table analysis can be statistically compared using bootstrap or jackknife methods 25,27 . Thus, life table analysis is a very effective tool for comparing the fitness of insects under different conditions. However, life table analysis of N. stenoferus has rarely been done.
Our goal in this study was to find a suitable diet for mass-rearing N. stenoferus and to better understand its biology. We compared its life history characteristics using life table analysis for groups reared using three diets: (1) aphids only (M. persicae), (2) moth eggs only (E. kuehniella), and (3) a mixed diet of aphids and moth eggs.
Adult females. There were significant differences among the diet treatments in the life history characteristics of adult females of N. stenoferus ( Table 2). The preoviposition period was significantly shorter in the group fed a mixed diet than in aphid-only or moth egg-only group (F = 16.11, df = 2, 17; P < 0.001). The oviposition periods varied from 5.7 to 41.6 days among the treatments, but without statistical significance (χ 2 = 5.85, df = 2, P = 0.054). The postoviposition period in the group fed the aphid-only diet was significantly longer than in moth egg-only group (1.4 days) (χ 2 = 7.50, df = 2, P = 0.024). There was no significant difference in female adult longevity among the diets tested (F = 1.76, df = 2, 25; P = 0.193). The total fecundity per female of bugs fed the mixed diet (aphids + moth eggs) was significantly higher than in groups fed the other two diets (F = 14.75, df = 2, 25; P < 0.001).

Discussion
In this study, we found significant effects of different food sources on the life history characteristics of N. stenoferus. Moth eggs alone did provide sufficient nutrients for both insect development and adult oviposition by N. stenoferus, but use of the aphid-only diet yielded sterile, or nearly sterile, adults. The mean fecundity in insects reared on the mixed diet was 87 times higher than bugs reared on the aphid-only diet. However, the fecundity of bugs reared on the moth egg diet was not significantly different from the group reared on aphid-only diet probably due to the very low number of ovipositing females obtained. Furthermore, the aphid-only diet lowered adult fitness of N. stenoferus by prolonging both the pre-and post-oviposition periods. Aphids are known to be low-quality foods for generalist predators, likely due to the presence of toxins or feeding deterrents 24 . However, in our study, there were no negative effects of an aphid-only diet on the development of the immature stage of N. stenoferus, and, consequently, we surmise that low adult fertility might be due to a nutritional deficiency in the aphid-only diet. Both protein and lipid diet components are known to significantly affect insect fecundity [28][29][30] . Proteins and lipids comprise 51.0 and 33.6% of the dry mass of Ephestia eggs 31 . In contrast, aphid-only diets have a lower lipid content than Ephestia eggs [28][29][30] . Different dietary needs for development in the immature stage versus reproduction in the adult stage are well known 32,33 . Different food exploitation patterns between immature and adult stages have frequently been reported in predatory mites. For example, immature stages of Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) can consume thrips and complete their stage's development despite a lower survival rate of this food 34 . However, the adult P. persimilis was known to rarely consume thrips as food 34 . Thus, a M. persicae-only diet might be suitable for the development of N. stenoferus nymphs but not suitable for adult maturation is consistent with other systems. However, there was a synergistic effect of aphid and moth egg diets for the life history characteristics of N. stenoferus in both the immature and adult stages. Numerous studies have reported the advantages of a mixed diet on insect fitness [35][36][37] . However, the noteworthy finding in our study is that the diet on which N. stenoferus completed its nymphal development (but yielded sterile adults) had a synergistic effect when mixed with other diet. Toft et al. 30 , in which Ephestia eggs and/or Rhopalosiphum padi (L.) aphids were used as food for Orius majusculus Reuter (Hemiptera: Anthocoridae), found lower fecundity in the aphid diet than in Ephestia egg diet. However, unlike our study, there was no synergistic effect on O. majusculus' fecundity from a mixed diet. In contrast, we found that the fecundity of N. stenoferus fed a mixed diet of aphids and moth eggs more than doubled compared to the moth eggs-only diet. The developmental period of the immature stage and the preoviposition period of N. stenoferus were also significantly shorter for bugs reared on the mixed diet. Furthermore, the mixed diet resulted in insects with a higher intrinsic rate of increase than did the other two treatments. This is the first finding of such contrasting contributions of diets on the development and reproduction of Hemiptera as far as we know.
Zoophytophagous hemipteran predators facultatively consume plant sap to obtain nutrients and water, but they can complete their life cycles only by feeding on animal prey such as insects or mites, as shown for N. stenoferus in this study [38][39][40][41][42]    Even though M. persicae alone is not a complete food source for N. stenoferus, it might be an error to regard this predator as an ineffective biological control agent against M. persicae or other aphid species. A diet that improves a predator's fitness does not necessarily engender a higher preference for that diet compared to others, even ones with intrinsically poorer nutrient profiles 30 . In Toft et al. 30 , O. majusculus, when reared on Ephestia eggs only or on a mixed diet of eggs and aphids, still preferred the poorer quality aphid-only diet. However, this predator, when reared on an aphid-only diet showed no preference between Ephestia eggs and aphids. Even though a diet might not be able to provide all the nutrients a predator might need, if the diet has the essential nutrients such as vitamins and amino acids, the predator may still consume the diet to prevent nutrient deficiency 46 . Unlike laboratory conditions, where the type of diet is artificially restricted, under natural conditions predators have opportunities to exploit mixed host resources efficiently for the best fitness gain 46 . The natural prey of N. stenoferus are known to be moth eggs or larvae, spider mites, and aphids 3 ; and N. stenoferus should maximize its fitness by exploiting mixed diets in nature. However, in agricultural areas, especially in greenhouses, a predator's diet choices may be limited by the simplification of the agroecosystem 47,48 . In predator-based augmentative biological control programs against aphid species in greenhouses, supplemental provision of Ephestia eggs might improve control 49 . Also, it may be possible to plant chrysanthemums in greenhouses as banker plants for N. stenoferus because this predator can complete its life cycle on chrysanthemums alone 1 . However, in open grassy fields, where N. stenoferus is an indigenous predator 2,3 , the species would be appropriate for conservation biological control 50,51 . Building up refuges with chrysanthemum plants can be suitable to enhance this predator than probably food spraying of Ephestia eggs, which can be used by other antagonists such as ants protecting aphids 52,53 .
In conclusion, the overall fitness of N. stenoferus was higher on a diet of E. kuehniella eggs than a diet of M. persicae, and a mixed diet of M. persicae and E. kuehniella eggs had a synergistic effect on the fitness of N. stenoferus in both the immature and adult stages. Therefore, we propose that M. persicae can be a supplementary food source for the mass-rearing of N. stenoferus, with the eggs of E. kuehniella the primary food source. Further studies might be needed on the nutritional composition of both E. kuehniella eggs and M. persicae to identify essential nutrients that might be responsible for the better fitness of N. stenoferus. To establish an efficient mass-rearing system for N. stenoferus, studies on the effects of environmental conditions such as temperature and humidity on N. stenoferus would also be needed. Moreover, as the use of Ephestia eggs for mass breeding of N. stenoferus may become expensive in certain regions or under specific conditions, additional research might be necessary to investigate the feasibility of utilizing relatively inexpensive diets such as brine shrimp eggs 54 .

Methods
Food sources. The eggs of E. kuehniella were used to feed our laboratory colony of N. stenoferus. Combinations of both E. kuehniella eggs and M. persicae (nymphs and adults mixed) were tested for the diet suitability. Both eggs and aphids were purchased from the Osang Kinsect, Namyangju, Korea. The eggs of E. kuehniella were frozen in the bottles received and taken out and used whenever necessary. Seedlings (about 10 cm height) of Chinese cabbage (B. rapa subsp. pekinensis) were purchased from the market in Andong, Korea, and planted in each pot (10 cm × 9.7 cm; diameter × height) and kept at 27 ℃, 60-80% RH, and a 16: Laboratory rearing of Nabis stenoferus. Nabis stenoferus was obtained from the Gyeonggi-do Agricultural Research and Extension Service in Hwaseong, Korea, and bugs were reared individually from egg to adult in Petri dishes (35 mm × 10 mm; diameter × height; SPL Life Science, Pocheon, Korea) at 27 ℃, 60-80% RH, and a 16:8 (L:D) h photoperiod in an incubator. Eggs of E. kuehniella, attached on parchment paper (1 × 1 cm), were provided as food for N. stenoferus rearing, and the egg papers were replaced daily. A piece of water-saturated cotton (0.8 × 0.8 × 0.8 cm) was placed in the rearing Petri dishes to supply water and as an oviposition substrate. When N. stenoferus individuals became adults, a pair of predators were kept in the Petri dish for mating.
Life table experiments. The experiment on rearing diets was conducted at 27.9 ± 0.76 ℃, 50.1 ± 6.4% RH, and a 16:8 (L:D) h photoperiod in an incubator. Twenty individuals (as newly laid eggs) of N. stenoferus were used for each food treatment. To obtain newly laid N. stenoferus eggs, ten adult females were randomly collected from the rearing colony for each treatment. Each female was allowed to lay eggs on water-saturated cotton in a Petri dish ( www.nature.com/scientificreports/ into a larger Petri dish (90 mm × 15 mm; diameter × height; SPL Life Science) and held for egg hatch. The eggs developmental period was recorded. The newly emerged nymphs were randomly selected and placed individually into experimental Petri dishes (50 mm × 15 mm; diameter × height; SPL Life Science) containing water-saturated cotton and a Chinese cabbage leaf disc (50 mm diameter). Diets were provided in Petri dishes as three treatments: (1) aphids only, (2) moth eggs only, and (3) both aphids and moth eggs. Aphid numbers in each Petri dish with aphids were maintained at least 40 individuals daily. The daily supply of aphids might be sufficient for Nabis sp. to meet its feeding needs (about 14.4 aphids were consumed per day in a previous study 55 ) with no observable food shortage. For treatments with moth eggs, one parchment paper (1 × 1 cm 2 ) with several hundred eggs was present in each Petri dish and was replaced daily.
When nymphs molted to adults, individual females and males were paired and held in new experimental Petri dishes. When female could not be immediately paired due to discrepancy in the number of molted males, we used male adults collected from the rearing colony. In the adult stage, only the life history characteristics of females were assessed, according to Maia et al. 25,26 . Developmental times of each life stage, female adult longevity, and daily fecundity were observed daily until all females had died. Data analysis. The data from any individuals lost during the experiment were discarded before analysis.
Females that did not lay eggs were excluded from the calculation of mean oviposition periods. The developmental periods for nymphs, as well as the oviposition and postoviposition periods were compared among treatments using the Kruskal-Wallis test in PROC NPAR1WAY in SAS 56 because of the non-normal distribution of the data. The preoviposition periods, as well as female adult longevity and fecundity were compared among treatments by analysis of variance using PROC GLM in SAS 56 .
Life table analysis and jackknife estimation of population parameters were carried out using the R program 57 by referring to Maia et al. 25,26 . Age-specific survival rates ( l x ) and fecundity ( m x ) for each treatment were calculated using the following equations: where SURV is the survival rate from egg to adult, NSF x is the number of surviving females at age x , and NF is the initial number of females. NEGG x is the mean number of eggs laid at age x , and SR is the sex ratio of each treatment group. The population parameters were calculated by the following equations 25 .
The net reproductive rate ( R 0 ) The mean generation time ( T) The intrinsic rate of increase ( r m ) The finite rate of increase ( ) The population parameters such as net reproductive rate ( R 0 ), mean generation time ( T ), intrinsic rate of increase ( r m ), and finite rate of increase ( ) were compared by Tukey's studentized range test after jackknife estimation 25 . www.nature.com/scientificreports/